Perhaps the classic genetic manipulation in molecular biology is the cleavage of a circular vector with one or more restriction enzymes and the ligation of a selected insert into the linearized vector. Since the 1970s, when the pioneers of molecular biology first demonstrated such manipulation to be feasible, significant research effort has been invested in the development of improved vector systems (see discussion of vectors derived from plasmids in Ausubel et al., Current Protocols in Molecular Biology, Section II, 1.5.1-1.5.17, John Wiley & Sons, 1998, incorporated herein by reference).
To give but a few examples, plasmid vectors that replicate in different hosts, with different copy numbers, have been prepared (e.g., bacterial vectors designed to have either relaxed or stringent control of replication; yeast vectors with either a 2μ or centromeric replication origin, mammalian vectors containing viral [e.g., SV40 or BPV] origins of replication, etc.). Vectors have been engineered to allow ready detection of insertion events (e.g., by creation or disruption of a selectable or detectable marker), to direct high levels of expression of proteins encoded by inserted sequences (e.g., under the control of transcription, splicing, and/or translation signals active in a given host system), to generate gene fusions that allow analysis of expression of inserted sequences (e.g., by analysis of B-galactosidase, chloramphenicol transferase, luciferase, or green fluorescent protein activity, etc.), or to create fusion proteins with experimentally useful attributes (e.g., easy purification, desired cellular localization, etc.). Vectors have been designed that are particularly useful for determining the sequence of inserted fragments (e.g., by allowing easy production of single-stranded DNA), or for producing RNA (sense or antisense) from the inserted sequences. Most companies that sell molecular biology reagents include among their products vectors that they have developed to be particularly useful for designated applications (see, for example, catalogs provided by Amersham Pharmacia Biotech, Piscataway, N.J.; Promega Corporation, Madison, Wis.; Invitrogen Inc., Carlsbad, Calif.; Life Technologies, Inc., Rockville, Md.; New England Biolabs, Beverly, Mass.; Stratagene, Inc., La Jolla, Calif.).
Of course, the universe of genetic “vectors” is not limited to circular molecules derived from bacterial plasmids. Any nucleic acid molecule that includes sequences sufficient to direct in vivo or in vitro self-replication can be employed as a vector. Typically, such replication sequences include a replication origin that directs duplication of the vector sequence in a host system (typically a transformed cell). Alternatively, sequences that direct integration of the vector into another nucleic acid molecule that is present in and replicated by the relevant host system can be sufficient to achieve vector (and insert) replication.
Most vectors in use today are derived from naturally-occurring bacterial plasmids, bacteriophages, or other viruses. Some vectors contain features of more than one of these systems. Almost all of the commonly-used vectors contain one or more restriction sites designed for convenient insertion of fragments; most have at least one polylinker (see, for example, the vector database maintained at the URL vectorbd.atcg.com/vectordb/vector.html, the contents of which as of Jul. 19, 2000 are included herein as Appendix A).
Despite the broad availability of vectors from commercial and other sources, each one has features selected by the relevant manufacturer rather than the experimental user. It is not uncommon for a researcher to have to modify an available vector to suit his experimental needs, or alternatively to modify his experimental design to accommodate the available vectors. There remains a need for the development of techniques and reagents that would allow a researcher to readily design and assemble vector(s) appropriate to his experimental needs.